The Tyrosine Kinase Inhibitors Imatinib and Dasatinib Reduce

Published OnlineFirst March 11, 2015; DOI: 10.1158/1535-7163.MCT-14-0849

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics The Tyrosine Kinase Inhibitors Imatinib and Dasatinib Reduce Myeloid Suppressor Cells and Release Effector Lymphocyte Responses Lisa Christiansson1, Stina Soderlund€ 2,3, Sara Mangsbo1, Henrik Hjorth-Hansen4,5, Martin Hoglund€ 2,3, Berit Markevarn€ 6, Johan Richter7, Leif Stenke8, Satu Mustjoki9, Angelica Loskog1, and Ulla Olsson-Stromberg€ 2,3

Abstract

Immune escape mechanisms promote tumor progression (arginase I, myeloperoxidase, IL10) as well as the presence of and are hurdles of cancer immunotherapy. Removing immu- natural killer cells, T cells (na€ve/memory), and stimulatory nosuppressive cells before treatment can enhance efﬁcacy. cytokines (IL12, IFNg,MIG,IP10).Bothimatinibanddasatinib þ þ Tyrosine kinase inhibitors (TKI) may be of interest to combine decreased the presence of CD11b CD14 CD33 myeloid cells with immunotherapy, as it has been shown that the inhibitor as well as the inhibitory molecules and the remaining myeloid sunitinib reduces myeloid suppressor cells in patients with suppressor cells had an increased CD40 expression. Monocytes renal cell carcinoma and dasatinib promotes expansion also increased CD40 after therapy. Moreover, increased levels of of natural killer–like lymphocytes in chronic myeloid leukemia CD40, IL12, natural killer cells, and experienced T cells were (CML). In this study, the capacity of dasatinib and imatinib noted after TKI initiation. The presence of experienced T cells to reduce myeloid suppressor cells and to induce immunomo- wascorrelatedtoahigherIFNg and MIG plasma concentration. dulation in vivo was investigated ex vivo.SamplesfromCML Taken together, the results demonstrate that both imatinib and patients treated with imatinib (n ¼ 18) or dasatinib (n ¼ 14) dasatinib tilted the immunosuppressive CML tumor milieu within a Nordic clinical trial (clinicalTrials.gov identiﬁer: towards promoting immune stimulation. Hence, imatinib and NCT00852566) were investigated for the presence of dasatinib may be of interest to combine with cancer immuno- þ þ CD11b CD14 CD33 myeloidcellsandinhibitorymolecules therapy. Mol Cancer Ther; 14(5); 1181–91. 2015 AACR.

Introduction immune system. It is now recognized that tumor cells avoid destruction by natural killer (NK) cells and T cells by releasing Immunotherapy of cancer has been in the limelight during the immunosuppressive molecules as well as molecules that pro- past few years due to the success of, for example, immunomod- mote the differentiation of myeloid suppressor cells and regu- ulatory antibodies such as ipilimumab (anti-CTLA-4) for malig- latory T cells (Treg). Myeloid suppressor cells are a heteroge- nant melanoma (1). Intense research in tumor immunology has neous group of myeloid cells that are increased in most cancer revealed the interplay between tumor cells, its stroma, and the patients (2). They utilize different mechanisms for suppressing immune responses, including upregulation of the enzyme Argi- 1Department of Immunology, Genetics and Pathology, Science for Life nase 1 (Arg1) leading to inhibition of T cells and other immune Laboratories, Uppsala University, Uppsala, Sweden. 2Department cells (3, 4). Moreover, myeloid suppressor cells can affect the of Medical Sciences, Uppsala University, Uppsala, Sweden. 3Section immune system by induction or recruitment of Tregs (5). Tregs of Hematology, Uppsala University Hospital, Uppsala, Sweden. 4Department of Hematology, St. Olav's Hospital, Trondheim, Norway. modulate the immune system by inhibition of effector T cells as 5Department of Cancer Research and Molecular Medicine, Norwegian well as NK cells, dendritic cells, and B cells (6). Immune evasion University of Science and Technology (NTNU) Trondheim, Norway. hampers antitumor immune reactions and is therefore since 6Department of Hematology, Norrland University Hospital, Umea , 2011 recognized as one of the hallmarks of cancer (7). Thus, Sweden. 7Department of Hematology and Coagulation, Skane University Hospital, Lund, Sweden. 8Department of Hematology, Kar- immune evasion strategies need to be considered during the olinska University Hospital and Karolinska Institute, Stockholm, Swe- development of immunotherapy. 9 den. Hematology Research Unit Helsinki, Department of Medicine, Most immunotherapies in clinical trials are currently used Division of Hematology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland. together with a preconditioning strategy to reduce the level of suppressive immune cells. For example, cyclophosphamide given Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). before treatment or metronomic during an extended period of time reduces the presence of Tregs (8). The tyrosine kinase € A. Loskog and U. Olsson-Stromberg contributed equally to this article. inhibitor (TKI) sunitinib used for patients with renal cell carci- Corresponding Author: Angelica Loskog, Uppsala University, Rudbeck Labo- noma was primarily used because of its capacity to inhibit VEGF € ratory C11, Dag Hammarskjolds v 20, Uppsala 75185, Sweden. Phone: 467-3537- signaling but it was soon recognized that hampering myeloid 7161; Fax: 461-8611-0222; E-mail: [email protected] suppressor cells was part of the mechanism of action (9). This doi: 10.1158/1535-7163.MCT-14-0849 inhibition has been linked to the suppression of STAT-3 signaling 2015 American Association for Cancer Research. (10) a feature shared also with other TKIs such as imatinib and

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dasatinib (11, 12). TKIs have been used to treat patients with a-CD40-FITC (clone: HB-14). Some patients and control subjects þ þ chronic myeloid leukemia (CML), as they target the constitutively had very low levels of CD11b CD14 CD33 myeloid cells, þ active tyrosine kinase bcr/abl present only in the CML tumor cells. thus, for these individuals CD40 expression on the CD11b þ The TKIs imatinib and dasatinib are both very effective for CD14 CD33 myeloid cells could not be analyzed. NK cells CML treatment and are well tolerated but in some patients, were stained with a-CD3-FITC and a-CD56/CD16-PE NK cell inflammation of unknown origin arises, implicating an effect on cocktail (clones: UCHT-1 and 3G8/MEM-188) and were defined þ the immune system (13). The high response rate is usually as CD3 CD56/CD16 cells. To exclude dead cells from the þ þ attributed to a direct effect of TKIs on the malignant cells. analysis of CD11b CD14 CD33 myeloid cells and NK cells, However, both in vitro (14–16) and in vivo (17–19) studies show cells were stained with the LIVE/DEAD Fixable Aqua Dead Cell that TKIs can modulate cells of the immune system, possibly Stain Kit, for 405 nm excitation (Life Technologies), according to affecting antitumor immunity of treated patients. the manufacturer's instructions. Cells that excluded the fluores- In this article, we sought to investigate the capacity of imatinib cent dye were considered live cells. For staining of different T-cell and dasatinib to reduce myeloid suppressor cells and their sup- phenotypes, a-CD3-FITC (clone: UCHT-1), a-CD8-APC (clone: pressive mediators in vivo to further understand their mechanism SK-1, BD Biosciences), a-CD45RA-APC/Cy7 (clone: HI-100), and þ of action in CML as well as to validate their capacity to function as a-CCR7-PerCP/Cy5.5 (clone: G043H7) were used. CD8 na€ve þ þ þ þ þ a preconditioning regimen prior, or during, immunotherapy of T cells were identified as CD3 CD8 CD45RA CCR7 , CD8 þ þ cancer. memory T cells were identified as CD3 CD8 CD45RA .To exclude dead cells from T-cell phenotype staining, 50 ng 406- Materials and Methods diamidino-2-phenylindole, dihydrochloride (DAPI, Life Tech- Patient and control subject samples nologies) was added to the tubes immediately before acquisition Samples from 32 patients were obtained from the "Random- in the flow cytometer. Cells that did not include DAPI were ized Study Comparing the Effect of Dasatinib and Imatinib considered live cells. Tregs were stained by the surface markers on Malignant Stem Cells in Chronic Myeloid Leukemia a-CD3-PerCP (clone: UCHT-1), a-CD4-FITC (clone: OKT-4), (NordCML006)" (clinicalTrials.gov identifier: NCT00852566). a–CD127-PE (clone: HIL-7R-M21, BD Biosciences) then the In this open labeled study, newly diagnosed chronic phase CML cells were fixed and permeabilized with the FOXP3 Fix/Perm patients were randomized to a starting dose of 100 mg dasatinib buffer set according to the instructions from the manufacturer once daily or 400 mg imatinib once daily. Eighteen patients (BioLegend). After permeabilization and blocking with normal treated with imatinib and 14 patients treated with dasatinib were mouse serum (Cedarlane), intracellular FoxP3 was stained with an a-FoxP3-Alexa Fluor 647 antibody (clone: 206D). Tregs analyzed for immune escape mechanisms and related markers. þ þ þ Plasma samples were taken at inclusion (pre, n ¼ 27), at one (n ¼ were defined as CD3 CD4 CD127 FoxP3 cells. The isotype 23), three (n ¼ 13), and six (n ¼ 23) months after initiation of the control antibodies IgG1-PE (clone: MOPC-21), IgG1-APC (clone: treatment (PTI). PBMCs were also taken before treatment (n ¼ 30) MOPC-21), IgG2b-brilliant violet 421 (clone: MPC-11), IgG1- and after 1 (n ¼ 31) and 6 (n ¼ 30) months PTI. Samples were FITC (clone: MOPC-21), and IgG1-Alexa Fluor 647 (clone: prepared and cryopreserved at the different study sites and cryo- MOPC-21) were used to identify background staining. All specific preserved samples were shipped to Uppsala, Sweden. The study antibodies and isotype controls were from BioLegend unless was performed according to the declaration of Helsinki and all otherwise stated. Cells were analyzed on LSRII (BD Biosciences) patients gave their written informed consent. The clinical study, and data was evaluated in Flow Jo software from Tree star. Before including this spin-off study, was approved by the medical gating of specific cell subsets, dead cells and duplets were removed products agencies as well as the regional research ethics commit- by gating (see Supplementary Figs. S1–S3). tees in the participating countries. PBMCs were obtained by Ficoll gradient separation (GE Healthcare). All samples had been cryo- Plasma analyses preserved before analysis. Plasma samples were investigated for the presence of Arg1 using an ELISA from Hycult Biotech according to the manufacturer's Flow cytometry instructions. Arg1 can be released from dying erythrocytes. Hence, Cryopreserved CML PBMCs from pre, 1, and 6 months PTI as to exclude the contribution of Arg1 from dying erythrocytes, well as cryopreserved PBMCs from control subjects were thawed hemolytic plasma samples were excluded from the analysis. IFNg in Iscove's modified Dulbecco's medium (IMDM) supplemented and IL10 were measured by proinflammatory 9-plex Ultra-Sen- with 10% human serum albumin and 1% penicillin/streptomy- sitive kit from Meso Scale Discovery Inc. MIG and IP10 were cin. IMDM and penicillin/streptomycin were from Life Technol- analyzed by a service by Myriad RBM Inc using a custom human ogies. Human serum albumin was from Octapharma AB. To MAP multiplex panel, whereas soluble CD40L, myeloid peroxi- remove clumps of dead cells after thawing and washing in dase (MPO), and IL12 were analyzed by a service by Olink 9696 supplemented IMDM medium, cells were run through a prese- Bioscience AB, using the ProSeek Multiplex Oncology . paration filter (Miltenyi Biotech). Before staining for flow cyto- ELISA, Meso Scale, and Myriad analyses gave concentrations of metry, unspecific binding was blocked by adding Fc-receptor analytes per mL plasma without correlation to plasma proteins. blocking reagent (Miltenyi Biotec). To identify different popula- Olink Bioscience AB's assay detects relative values to compare tions of cells, extra- and intracellular staining were performed. differences among groups. Blocked cells were stained with specific antibodies and corre- þ þ sponding isotype controls. To identify CD11b CD14 CD33 Proliferation assay myeloid cells, the cells were stained with a-CD11b-PE/Cy7 The suppressive effect of CML myeloid cells on healthy (clone: ICRF 44), a-CD14-Alexa Fluor 700 (clone: M5E2), and donor T-cell proliferation was determined in a 3H-thymidine a-CD33-APC (clone: WM-53). These cells were also stained with assay. Briefly, leukocytes were collected from three newly

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Table 1. Patient characteristics Sokal EUTOS WBC BCR-ABL MMR Dose Adverse Conc. Pat. no. Age Gender group group (106) Treatment >10%a month 12 intensity (%) events, grade med. 1 43 M LR LR 28 Ima No No 100 Leukopenia, 2 No 2 55 F LR LR 75 Ima No No 96 Leukopenia, 3 G-CSF 3 60 F IR LR 32 Dasa No Yes 100 Anemia, 2 No 4 45 M LR LR 32 Dasa No Yes 100 Leukopenia, 2 Anemia, 1 No 5 51 F IR HR 40 Dasa No Yes 100 No No 6 62 M LR LR 26 Ima No Yes 99 No No 7 40 F LR LR 11 Dasa No Yes 100 Thrombopenia, 2 No 8 66 M LR LR 30 Ima No No 100 Leukopenia, 2 No thrombopenia, 2 9 39 M LR LR 18 Ima No Yes 100 No No 10 71 F IR HR 14 Dasa No Yes 100 Leukopenia, 2 No thrombopenia, 2 11 61 F LR LR 20 Dasa No Yes 100 Anemia, 1 NSAID 12 52 F IR LR 109 Ima No No 100 Leukopenia, 3 G-CSF 13 60 M HR LR 288 Ima Yes No 100 Leukopenia, 2 G-CSF thrombopenia, 4 14 63 M LR LR 22 Ima No No 100 No No 15 44 F IR LR 164 Dasa No Yes 100 Leukopenia, 2 No 16 45 F LR LR 39 Dasa No Yes 100 No No 17 73 F IR LR 45 Ima No Yes 100 No No 18 47 F LR LR 46 Dasa No Yes 100 Neutropenia, 3 G-CSF 19 57 F LR LR 38 Ima No No 100 No No 20 51 F LR LR 74 Ima No Yes 100 No NSAID 21 71 M IR LR 37 Ima No Yes 100 Leukopenia, 2 No 22 58 M LR LR 43 Dasa No Missingb 64 Pleural effusion, 2 G-CSF, steroid 23 37 M LR LR 165 Ima Yes No 100 Neutropenia, 2 No 24 61 M HR HR 336 Dasa No Yes 100 No No 25 63 F HR LR 20 Dasa No Yes 100 No No 26 67 F HR HR 70 Ima No Yes 92 Leukopenia, 2 No 27 43 M LR LR 154 Ima No No 100 No No 28 28 F IR LR 270 Dasa No No 95 No No 29 45 F LR LR 25 Dasa No Yes 96 Leukopenia, 3 No 30 55 M LR LR 65 Ima No Yes 100 No No 31 59 F IR LR 97 Ima Yes No 100 No No 32 43 M LR LR 110 Ima Yes No 100 No No NOTE: Sokal group: LR, low risk (score <0.8); IR, intermediate risk (score 0-8–1-2); HR, high risk (score >1.2). EUTOS group: LR, low risk (score 87); HR, high risk (score >87). Abbreviations: Ima, imatinib; Dasa, dasatinib. aBCR-ABL >10% at month 3; MMR, major molecular response; BCR-ABL is 0.1%; grading of adverse events is according to CTCAE. bMMR at 6 months. diagnosed CML patients at three occasions. Because of poor unpaired groups while Wilcoxon matched-pairs signed rank test viability of CML cells after freeze/thaw cycles, the cells were was used to determine differences between pre and PTI samples. treated with a dead cell removal kit that reduce the number of One-way ANOVA was used to calculate differences when more dead cells before further analyses (Miltenyi Biotec). The mye- than two samples were compared and those groups were then post þ loid fraction was selected by CD3 sorting using MACS beads tested using Wilcoxon for matched pairs. For correlation analysis, and collecting the ﬂow through CD3 fraction (Miltenyi Bio- the nonparametric Spearman rank correlation was assessed. All tec). The myeloid CD3 fraction was irradiated with 40 Gy and statistical analyses were made with Prism Software (GraphPad used as allogeneic stimulation in a T-cell proliferation assay. Software Inc.). þ Allogeneic healthy donor CD3 cells were sorted from PBMCs by MACS beads and were then mixed with the CML myeloid CD3 fraction in a 1:1 ratio and cultured in triplicates in a Results round-bottom 96-well plate. 3H-thymidine (1 uCi; PerkinEl- Patient characteristics mer was added to the wells and the cells were cultured for 3 A summary of patients' baseline characteristics is shown days before they were harvested to ﬁlters and counts per minute in Table I. The median age of the patients was 55 years with the (cpm) were measured by a Wallac b-counter (PerkinElmer Life dasatinib patients being slightly younger. In the dasatinib-treated Science). As a positive proliferation control in each of the three group, more patients were in the Sokal and Eutos intermediate experiments, the myeloid CD3 fraction from healthy donors and high risk. Only 5 patients (patient number 13, 15, 24, 28, þ wasusedtostimulatethesameallogeneicCD3 Tcells. and 31) had spleen enlargement at diagnosis. The dose intensity was generally high (as seen in Table 1) and pauses in treatment Statistical analysis were due to hematologic toxicity in all cases but patient no 22 The Student t test with Welsh correction for possibly unequal who developed pleural effusion on dasatinib treatment. After variances was used to determine statistical differences between 12 months of TKI treatment, 59% of the patients had a major

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Figure 1. þ þ CD11b CD14 CD33 myeloid cells after TKI treatment. Myeloid cells (CD3 PBMCs) from 3 CML patients and 3 healthy controls (HC) were irradiated and cocultured with þ allogeneic healthy donor CD3 T cells in three separate experiments. Proliferation was measured using thymidine incorporation. A, the fold change of T cells stimulated with allogeneic healthy donor myeloid cells compared with allogeneic CML- derived myeloid cells is shown. PBMCs from CML patients pre- and 6 months after TKI therapy initiation (PTI) were analyzed by flow cytometry. B, the difference in myeloid suppressor cells pre and PTI is shown while in C the levels in patients treated with imatinib (square) and dasatinib (dot), respectively, are shown. Statistical significance was calculated using the Wilcoxon test for nonparametric, þ paired samples. The CD11b CD14 CD33 myeloid cells were correlated to the leucocyte count before TKI therapy (D), or compared between in the MMR and no MMR groups at 12 months (E). Statistical significance was calculated using the Spearman correlation test for nonparametric testing. P values <0.05 were considered significant. Error bars, median with interquartile range.

molecular response (MMR). In-depth analysis of the clinical the allogeneic T cells showed less proliferative capacity when results have been published previously (20). cultured with myeloid cells from CML patients (Fig. 1A). As TKIs þ may reduce the presence of such cells, the level of CD11b þ þ þ The level of CD11b CD14 CD33 myeloid cells decreased CD14 CD33 myeloid cells in CML patients pre- and postinduc- after TKI treatment tion of imatinib or dasatinib therapy was investigated. The medi- þ þ We have previously shown that CD11b CD14 CD33 mye- an level of these cells in PBMCs before treatment was 3.2% (range loid suppressor cells are increased in Sokal high-risk CML patients 0.2–11.3%, Fig. 1B, gating strategy in Supplementary Fig. S1). þ þ (21). To demonstrate the suppressive capacity of myeloid cells in After 6 months of treatment, the levels of CD11b CD14 CD33 CML patients, we depleted the CD3 lymphocytes from three myeloid cells were signiﬁcantly decreased (P ¼ 0.0034). In Fig. 1C, newly diagnosed patients and three healthy controls and tested the responses to either imatinib or dasatinib are displayed for each the ability of the remaining myeloid cell population to inhibit patient. There was no difference in this cell population between allogeneic T-cell proliferation. Allogeneic T cells expanded vigor- imatinib- and dasatinib-treated patients. In solid tumors, the ously upon coculture with healthy donor myeloid cells, whereas number of circulating myeloid suppressor cells correlates

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Figure 2. Arg1 and MPO after TKI treatment. Expression of cytokines connected to myeloid suppressor cells. Plasma samples at pre and 6 months PTI were analyzed for Arg1 using ELISA (A) where after the Arg1 level pretreatment was correlated to CD11bþCD14CD33þ myeloid cells at base line (B). Plasma samples were further analyzed for MPO (C), GM-CSF (D), IL6 (E), TGFb (F), VEGF (G), and IL8 (H) before and at 3 months PTI using ProSeek (C–G) and MesoScale (H). NPX, normalized protein expression. Statistical signiﬁcances were calculated using Wilcoxon test for paired samples. P values <0.05 were considered signiﬁcant. Error bars, median with interquartile range.

with metastatic tumor burden and clinical cancer stage (22). of TKI therapy, the median Arg1 concentration in patient Likewise, in CML patients, the leukocyte count (reflecting plasma was significantly decreased (Fig. 2A; P ¼ 0.0001). The the tumor burden of a patient) before treatment correlated with concentration of Arg1 before treatment was correlated to the þ þ þ þ the CD11b CD14 CD33 myeloid cells (Spearman r: 0.6713, number of CD11b CD14 CD33 myeloid cells (Fig. 2B; Spear- P < 0.0001; Fig. 1D). However, there was no correlation between man r: 0.5238, P ¼ 0.005). On a fraction of the patients (n ¼ 13, MMR values and MDSCs, and no significant difference could be dasatinib n ¼ 5; imatinib n ¼ 8), the MPO concentration before calculated when comparing the MDSC levels in the MMR group and 3 months PTI was measured. MPO was significantly decreased versus no MMR at 12 months (Fig. 1E). Arg1 and MPO are after treatment initiation (Fig. 2C; P ¼ 0.0002). Furthermore, suppressor molecules connected to myeloid cells. After 6 months growth factors connected to the development of MDSCs such as

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Figure 3. Tregs and CD40 expression on cells in peripheral blood of CML patients after TKI treatment. A, CD40 expression was determined on myeloid suppressor cells (CD11bþCD14CD33þ) and on monocytes (CD14þ;B)usingflow cytometry in CML patients and healthy controls pre and at 6 months PTI. C, the plasma concentration of soluble CD40L was evaluated using ProSeek. NPX refers to normalized protein expression. D, Tregs (CD3þCD4þCD127FoxP3þ) were quantified using flow cytometry pre and PTI in CML patients and healthy controls. The plasma concentration of IL10 (E) and IL12 (F) was determined using MesoScale 9-plex kit and Proseek, respectively. Statistical significances were calculated using Wilcoxon test for paired samples and Student t test with Welsh correction for unpaired calculations (e.g., comparison to healthy controls). P values <0.05 were considered significant. Error bars, median with interquartile range.

þ þ þ GM-CSF, IL6, TGFb, VEGF were not significantly different (Fig. Tregs (CD3 CD4 FoxP3 CD127 ) were increased 6 months 2D–G). GM-CSF was not detected in most patients. However, IL8 after initiation of TKI therapy (P ¼ 0.0073) but the percentage was significantly enhanced by TKI therapy (Fig. 2H). of Tregs in treated patients was not significantly different from healthy controls (Fig. 3D). Instead, the Treg-associated cytokine þ þ CD40 expression on CD11b CD14 CD33 myeloid cells IL10 was decreased in patient plasma 3 months posttreatment To investigate the status of the myeloid cells in CML patients (n ¼ 13, P ¼ 0.0266; Fig. 3E), whereas the concentration of the Th1 after TKI therapy, the immunomodulatory receptor CD40 was effector cell stimulator IL12 was increased (n ¼ 13, P ¼ evaluated. Stimulation of myeloid suppressor cells with soluble 0.0081; Fig. 3F). IL12 is released from activated myeloid cells monomeric CD40L has recently shown to increase their suppres- such as mature dendritic cells (DC) that promote T- and NK cell þ þ þ sive capacity (23). Interestingly, before therapy, the CD40 expres- activation indicating that the CD3 CD4 FoxP3 CD127 cells sion was significantly lower on both myeloid suppressor cells may not reflect true Tregs but merely recently activated T cells that þ þ þ (CD11b CD14 CD33 ; P < 0.0001) and monocytes (CD14 ; P may also transiently express FoxP3. Collectively, these findings < 0.0001) compared with the CD40 levels in healthy individuals. point to immune activation rather than increased immunosup- However, the total number of myeloid suppressor cells is low in pression and the level of lymphocytes and lymphocyte-associated healthy individuals and this may reflect the high level of CD40. molecules were thus investigated. After initiating TKI therapy, CD40 increased on the myeloid suppressor cells (pre vs. post: P ¼ 0.0008) and on the monocytes Activation of Th1 effector cells and cytokines by TKI therapy (pre vs. post: P ¼ 0.0003; Fig. 3A and B). However, the level of It has previously been shown that NK cells in CML patients are sCD40L in plasma (n ¼ 13) was not affected by TKIs (Fig. 3C). activated after dasatinib therapy initiation (19). In our cohort of

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Figure 4. The number of NK cells and experienced T cells in CML patients. The level of NK cells (CD3 CD56/ CD16þ; A) and T cells (B: na€ve CD3þ CD8þCD45RAþCCR7þ;C: experienced CD3þCD8þCD45RA) was analyzed in CML patients before, at 1 month, and at 6 months PTI using flow cytometry. The ratio of T memory cells and myeloid suppressor cells (CD11bþCD14CD33þ) was calculated and displayed in D. Statistical significances were first calculated using one-way ANOVA for comparison of all three paired groups. This test showed that there was a difference between the groups NK cells and na€ve T cells. To calculate the exact difference between the pre and a specific time point Wilcoxon test for paired samples was used. For the analysis of experienced T cells, some time points were not analyzed by flow cytometry and, hence, the one-way ANOVA could not be performed. In the figure, the significant difference is only calculated using paired sample Wilcoxon test. P values <0.05 were considered significant. Error bars, median with interquartile range.

patients, the NK cells expanded and demonstrated a significant cells in our samples. Interestingly, high levels of MIG and IFNg PTI increase after 1 month but not at 6 months PTI (Fig. 4A; P ¼ correlated to high levels of experienced T cells in patients before 0.0243). This effect was seen in both imatinib- and dasatinib- treatment (Fig. 5D and E; P ¼ 0.0034 and P ¼ 00485, respectively) treated patients. Five of 6 patients with the highest NK cell levels and they tended to be correlated to the level of experienced T cells were in MMR at 12 months but the cohort is unfortunately too 1 month PTI (Fig. 5F and G). However, NK cells did not correlate þ small for statistical correlations to MMR. Na€ve CD8 T cells to the presence of experienced T cells or to the molecules IFNg and þ þ þ þ (CD3 CD8 CD45RA CCR7 ) were decreased after 1 month of MIG (Fig. 5H–J). TKI treatment (Fig. 4B; P ¼ 0.0050) and most patients also stayed at this lower level after 6 months of treatment. In contrast, þ þ experienced (effector and memory, CD3 CD8 CD45RA ) Discussion þ CD8 T cells were instead increased after 1 month of TKI treat- TKIs such as sunitinib decrease the level of myeloid suppressor ment initiation similarly to the NK cells (Fig. 4C; P ¼ 0.0107). cells in cancer and this may lead to enhanced antitumor immune Interestingly, the ratio of experienced T cells to myeloid suppres- responses. Therefore, TKIs may be of great interest to combine sor cells was increased after treatment initiation indicating a shift with active immunotherapy to increase efficacy. Indeed, sunitinib in the T cell:suppressor cell balance in favor of active T-cell increased the efficacy of peptide-pulsed dendritic cells in a murine responses (Fig. 4D). B16.OVA model (24). Other TKIs such as imatinib or dasatinib Because both NK cells and T cells release IFNg upon activation may share the same mechanisms to reduce suppressive cells and and killing of target cells, the levels of IFNg, the monokine may thus be interesting candidates for preconditioning regimens. induced by IFNg (MIG) and IFNg-induced protein 10 (IP10) Both imatinib and dasatinib have been used extensively in CML were investigated in a cohort of patients (n ¼ 13) 3 months after and are well tolerated for extended periods of time. Moreover, initiation of TKI therapy from plasma. IFNg was below detection they have been connected to immunologic actions. For example, limit for most patients before treatment but was detected in expansion of a population of large granular lymphocytes of T- or several of the patients after TKI therapy initiation and the increase NK cell origin in patients treated with dasatinib has been of IFNg was significant (Fig. 5A; P ¼ 0.0273). Likewise, MIG described (19). Expanded T cells were of both a/b and g/d type was increased PTI (Fig. 5B; P ¼ 0.0015), whereas IP10 was stable (25). This lymphocyte expansion was associated with good treat- (Fig. 5C). To investigate whether high MIG and IFNg levels PTI are ment responses, but also with side effects like pleuritis and colitis good markers for ongoing lymphocyte activity, we correlated the (19, 25). The cause of lymphocyte expansion remains unknown. MIG and IFNg levels to the presence of experienced T cells and NK Both a rapid decrease of tumor cells, and thereby a decrease in

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Figure 5. NK- and T-cell–associated cytokines in CML patients. Plasma from CML patients (n ¼ 13) was analyzed before and at 3 months PTI to determine the level of IFNg (A), MIG (B), and IP10 (C). IFNg was evaluated using an ultrasensitive 9-plex kit from MesoScale while MIG and IP10 were evaluated using a custom Myriad RBM kit. Statistical significances were calculated using Wilcoxon test for paired samples. MIG concentration PTI was correlated to experienced T cells pre and PTI (D and G) and IFNg concentration PTI was correlated to experienced T cells pre and PTI (E and F). NK cell levels PTI were then correlated to experienced T-cell levels PTI (H), to IFNg (I), and to MIG (J). Significant differences were calculated using Spearman correlation test for nonparametric samples. P values <0.05 were considered significant.

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potentially immunosuppressive cells, as the tumor cell per se may on Tregs after initiating TKI may give different results in another be immunosuppressive, as well as off-target effects on other type of cancer. For example, in patients with gastrointestinal tyrosine kinases present in immune cells may serve as explana- stromal tumors (GIST), Treg levels were decreased after initiation tions for the lymphocyte expansion. In a recent work, it was of imatinib therapy (34). However, the patients had received further demonstrated that dasatinib induced granzyme B in the imatinib during a median of 11.7 months, which is longer than expanded cells, demonstrating their ongoing activity (26). We our patients. In another study on GIST, imatinib was shown to have demonstrated previously that sunitinib upregulated increase M2 macrophages and thereby increasing IL10 (35), in CXCL10 and CXCL11 in tumor vessels which was accompanied our patients IL10 is decreased. Nevertheless, GIST patients have by up to 18-fold increased infiltration of T cells in a melanoma aC-KIT exon 11 mutation that may explain discrepancies to the model (27). Likewise, dasatinib was shown to enhance the effect effect of imatinib in CML. Alternatively, the effect of TKIs may be of immunotherapy in melanoma partly by upregulating CXCL9, depending on the ongoing immune profile at the start of treat- 10, and 11, as well as reducing the presence of both Tregs and ment. More studies to confirm the role of TKIs in different patient MDSCs (28). The exact mechanisms have been difficult to dissect groups are clearly warranted. Upregulation of CD40 was also as PBMCs including T cells lose viability or reduce their prolifer- noted on other myeloid cells such as monocytes in our study. ation upon in vitro culture with TKIs such as dasatinib (29, 30). It is CD40 is an important receptor on myeloid cells and upon ligation likely that the continuous dose of TKIs in culture media has a they commonly mature and increase their expression of costimu- negative effect on the immune cells that may not be seen in latory molecules and inflammatory cytokines including IL12 patients, as the dose is not constant. Furthermore, TKIs may affect which is important for activation of Th1 type of immune other cell types in vivo that give secondary immune activating responses as mentioned above (32). In the CML patients, IL12 effects that may override possible negative effects of TKIs. For was indeed increased after TKI therapy initiation. Hence, the example, inhibiting the MDSCs may release expansion of lym- upregulation of CD40 on myeloid cell populations in treated phocytes and the overall TKI effect may be stimulatory. There may patients may be of immunologic importance to release antitumor also be other not yet defined factors in vivo that protect the immune responses. This connection, however, needs to be exper- lymphocytes from the toxic effects of TKIs noted in vitro. In the imentally confirmed. present work, we therefore investigated the in vivo effect of We next investigated the presence of NK cells and T cells after imatinib and dasatinib on the immune system in patients with TKI therapy. We confirmed that NK cells were expanded in some CML. of the patients during TKI treatment and that there was no We have previously demonstrated that CML patients have an significant difference between imatinib or dasatinib therapy in þ increased level of myeloid suppressor cells defined as CD11b our cohorts. However, the increase was significant after one þ þ CD14 CD33 cells. This phenotype was seen on both CD34 months of TKI treatment and thereafter the number of NK cells and CD34 cells in the peripheral blood indicating that not only was reduced to normal levels in most patients. Similarly, effector healthy myeloid cells (CD34 ) but also a proportion of the and memory T cells were significantly increased at 1 month after þ þ leukemic myeloid cells (CD34 bcr/abl ) may have a suppressor TKI therapy initiation in some patients but at the 6 months cell phenotype (21). In the current study, we demonstrated sampling, the level was normalized. Hence, there may be an that both imatinib and dasatinib treatment efficiently decreased initial stimulatory effect on the immune system, as the numbers þ þ the suppressive peripheral CD11b CD14 CD33 myeloid of experienced T cells and NK cells increases, likely because of the cells. Simultaneously, suppressive molecules connected to mye- reduction of suppressive factors. Nevertheless, this effect is lost loid cells such as Arg1 and MPO were significantly decreased. with time, which may depend on the lack of incitements of Before TKI therapy, the patients had reduced level of CD40 on immune activation, as the tumor burden is decreased to unde- their immature myeloid cells but, interestingly, the remaining tectable levels in most patients. Most patients that had initial high þ þ CD11b CD14 CD33 myeloid cells upregulated CD40 on their levels of experienced T cells remained high during TKI therapy. surface after treatment initiation indicating that they may differ- The ratio of T memory cells to na€ve T cells was previously shown entiate or even mature. CD40/CD40L signaling has shown to to be higher in patients that remained in MMR after treatment improve the capacity of myeloid suppressor cells to induce Tregs discontinuation compared with both control subjects and to (31). There is a study demonstrating that stimulation of myeloid patients that relapsed after therapy discontinuation (36). Corre- suppressor cells with soluble monomeric CD40L resulted in an spondingly, in our study, the level of na€ve T cells was reduced in increased capacity to suppress T-cell proliferation (23) as opposed favor of effector and memory T cells. Moreover, IFNg was detected to the induction of Th1 immunity noted when dendritic cells are in patient plasma in some of the patients during TKI therapy stimulated via CD40L (32). In our patient cohort, there was no indicating active NK or T-cell responses. IFNg stimulates the difference in sCD40L levels. However, the level of Tregs was production of MIG (37) and this monokine was also increased significantly increased while its associated inhibitory molecule, after initiation of TKI therapy. However, IP10 that is also induced IL10, was decreased in most patients even if the overall presence of by IFNg was only increased in some patients in our cohort. As the IL10 in plasma was very low and the biologic role of these levels patients with high T-cell numbers did not simultaneously have are not determined. We cannot exclude that the increase in high NK cell numbers, it is possible that different patients respond þ FoxP3 cells reflected effector T cells that transiently express FoxP3 to TKI therapy either with T cells or NK cells dependent on factors even if we gated for the CD127 population. In a study by yet unknown. Balachandran and colleagues, imatinib was shown to decrease In conclusion, the results in our study demonstrate that both Tregs in an animal model (33), which further supported that the imatinib and dasatinib can reduce immune escape mechanisms þ increase of FoxP3 cells in our cohort post TKI may not reflect true by decreasing the number of myeloid suppressor cells and inhib- Tregs. The level of Tregs in CML patients before TKI therapy was itory cytokines Arg1, MPO, and IL10. Moreover, upregulation of not increased compared with healthy donors and, hence, the effect CD40 and IL12 production indicate ongoing adaptive immunity.

www.aacrjournals.org Mol Cancer Ther; 14(5) May 2015 1189

Christiansson et al.

NK cells or T cells expand during the first month of treatment Analysis and interpretation of data (e.g., statistical analysis, biostatistics, and IFNg andMIGcouldbedetectedinplasmaduringTKI computational analysis): L. Christiansson, S. Soderlund, A. Loskog, U. Olsson- therapy. Even if the most important mechanism of imatinib Stromberg Writing, review, and/or revision of the manuscript: L. Christiansson, and dasatinib therapy in CML is the direct effect on the tumor S. Soderlund, H. Hjorth-Hansen, M. Hoglund,€ J. Richter, L. Stenke, S. Mustjoki, cells by inhibiting the bcr/abl kinase, the long-term effect may A. Loskog, U. Olsson-Stromberg also be dependent on activated antitumor immune responses. Administrative, technical, or material support (i.e., reporting or organizing Moreover, because of the potent reduction of immune escape, data, constructing databases): L. Christiansson, S. Soderlund, S.M. Mangsbo, atthesametimeactivatinglymphocytes,andtherelativelymild H. Hjorth-Hansen, S. Mustjoki, U. Olsson-Stromberg adverse events due to imatinib or dasatinib, these TKIs may be Study supervision: S.M. Mangsbo, H. Hjorth-Hansen, A. Loskog, U. Olsson- Stromberg interesting options for preconditioning cancer patients before immunotherapy. For example, combining TKI with T-cell ther- in vivo apy may allow for a better persistence of the infused Acknowledgments T cells. Furthermore, active immunotherapy such as peptide BioVis at SciLifeLab in Uppsala is acknowledged for providing equipment for vaccinations may be boosted by TKIs due to the reduction of flow cytometry analysis. The authors would like to thank personnel at the suppressive factors. Hematology Research Unit in Helsinki and at Meso Scale Discovery Inc for technical assistance during sample analysis as well as Olink Bioscience AB for Disclosure of Potential Conflicts of Interest providing for plasma analyses and technical support of data management. The H. Hjorth-Hansen reports receiving commercial research grants from authors also thank Emma Svensson and Jessica Wenthe at Uppsala University Bristol-Myers Squibb, Merck, and Novartis, and is a consultant/advisory for technical assistance. board member of Bristol-Myers Squibb and Novartis. J. Richter reports receiving commercial research grants from Bristol-Myers Squibb, Merck, and Novartis and received speakers bureau honoraria from Novartis and Grant Support Bristol-Myers Squibb. S. Mustjoki reports receiving commercial research The European LeukemiaNet and the Nordic CML study group supported this grants from Pfizer, Novartis, Bristol-Myers Squibb, and Merck; received study by grants to Dr U. Olsson-Stromberg,€ and the Medical Faculty at Uppsala speakers bureau honoraria from Bristol-Myers Squibb and Novartis; and is University supported this study by grants to Dr A. Loskog. Olink Biosciences AB a consultant/advisory board member for Bristol-Myers Squibb and Novartis. provided support for plasma analyses (to A. Loskog). The Nordic CML Study A. Loskog reports receiving other commercial research support from Olink Group (represented by H. Hjorth-Hansen, M. Hoglund,€ B. Markev€arn, J. Richter, Biosciences AB and is a consultant/advisory board member for NEXTTOBE L. Stenke, S. Mustjoki, and U. Olsson Stromberg)€ received research funding from AB. U. Olsson-Stromberg reports receiving a commercial research grants Bristol-Myers Squibb for the clinical trial (NordCML006) presented in this from Bristol-Myers Squibb, Novartis, and Merck. No potential conflicts of article. interest were disclosed by the other authors. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Authors' Contributions advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Conception and design: L. Christiansson, A. Loskog, U. Olsson-Stromberg this fact. Development of methodology: L. Christiansson, U. Olsson-Stromberg Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Christiansson, S.M. Mangsbo, H. Hjorth-Hansen, Received October 1, 2014; revised February 4, 2015; accepted February 17, M. Hoglund,€ B. Markev€arn, J. Richter, L. Stenke, S. Mustjoki, U. Olsson-Stromberg 2015; published OnlineFirst March 11, 2015.

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www.aacrjournals.org Mol Cancer Ther; 14(5) May 2015 1191

The Tyrosine Kinase Inhibitors Imatinib and Dasatinib Reduce Myeloid Suppressor Cells and Release Effector Lymphocyte Responses

Lisa Christiansson, Stina Söderlund, Sara Mangsbo, et al.

Mol Cancer Ther 2015;14:1181-1191. Published OnlineFirst March 11, 2015.

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